Bifunctional polypeptides

ABSTRACT

A bifunctional polypeptide comprising a specific binding partner for a peptide-MHC epitope, such as an antibody or T cell receptor, and an immune effector, such as an antibody or a cytokine, the immune effector part being linked to the N-terminus of the peptide-MHC binding part.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 16/151,144, filed Oct. 3, 2018, which is a continuation of U.S.application Ser. No. 13/319,597, filed Apr. 5, 2012, now U.S. Pat. No.10,130,721, which is the National Stage of International Application No.PCT/GB2010/000988, filed May 19, 2010, which claims the benefit of andpriority to Great Britain Patent Application No. 0908613.3, filed May20, 2009, each of which is herein incorporated in its entirety byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 4, 2019, isnamed 42800US_CRF_SequenceListing.txt and is 23,605 bytes in size.

FIELD OF THE INVENTION

This invention relates to a bifunctional polypeptide comprising aspecific binding partner for a peptide-MHC epitope, such as an antibodyor a T cell receptor (“TCR”), and an immune effector, such as anantibody or a cytokine, the immune effector part being linked to theN-terminus of the peptide-MHC binding part.

BACKGROUND TO THE INVENTION

TCRs mediate the recognition of Specific Major HistocompatibilityComplex (MHC)-peptide complexes (“pMHC complexes”) which are presentedas epitopes on antigen presenting cells (APC), and TCRs mediate therecognition of such pMHC epitopes by T cells. As such TCRs are essentialto the functioning of the cellular arm of the immune system.

Antibodies are also known which specifically bind pMHC epitopespresented by antigen presenting cells (see for example: Neethling F A.et al., Vaccine (2008) 26 (25): 3092-102). There are antigen-bindingfragment (Fab) antibodies (see for example: Chames P. et al., Proc NatlAcad Sci USA (2000) 97 (14): 7969-74; Willemsen R A. et al., J Immunol(2005) 174 (12): 7853-8; Willemsen R. et al., Cytometry A (2008) 73(11): 1093-9) or single-chain antibody fragments (scFv) (see forexample: Denkberg G. et al., J Immunol (2003) 171 (5): 2197-207; MargetM. e al., Mol Immunol (2005) 42 (5): 643-9) which specifically bind pMHCepitopes.

The native TCR is a heterodimeric cell surface protein of theimmunoglobulin superfamily which is associated with invariant proteinsof the CD3 complex involved in mediating signal transduction. TCRs existin αβ and γδ forms, which are structurally similar but have quitedistinct anatomical locations and probably functions. The MHC class Iand class II ligands are also immunoglobulin superfamily proteins butare specialised for antigen presentation, with a highly polymorphicpeptide binding site which enables them to present a diverse array ofshort peptide fragments at the APC cell surface.

The extracellular portion of native heterodimeric αβ TCRs consist of twopolypeptides (the α and β chains) each of which has a membrane-proximalconstant domain, and a membrane-distal variable domain. Each of theconstant and variable domains includes an intra-chain disulfide bond.The variable domains contain the highly polymorphic loops analogous tothe complementarity determining regions (CDRs) of antibodies. CDR3 of αβTCRs interact with the peptide presented by MHC, and CDRs 1 and 2 of αβTCRs interact with the peptide and the MHC. The diversity of TCRsequences is generated via somatic rearrangement of linked variable (V),diversity (D), joining (J), and constant genes (C).

Functional a chain polypeptides are formed by rearranged V-J-C regions,whereas β chains consist of V-D-J-C regions. The extracellular constantdomain has a membrane proximal region and an immunoglobulin region.There is a single a chain constant domain, known as TRAC. The β chainconstant domain is composed of one of two different β constant domains,known as TRBC1 and TRBC2 (IMGT nomenclature). There are four amino acidchanges between these β constant domains. These changes are all withinexon 1 of TRBC1 and TRBC2: N₄K₅->K₅N₅ and F₃₇->Y (IMGT numbering,differences TRBC1->TRBC2), the final amino acid change between the twoTCR β chain constant regions being in exon 3 of TRBC1 and TRBC2: V₁->E.

A number of constructs have been devised to date for the production ofrecombinant TCRs. These constructs fall into two broad classes,single-chain TCRs and dimeric TCRs. Single-chain TCRs (scTCRs) areartificial constructs consisting of a single amino acid strand, whichlike native heterodimeric TCRs bind to MHC-peptide complexes. scTCRs canconsist of a combination of TCR α and β variable regions (Vα and Vβrespectively) and TCR α and β constant regions (Cα and Cβ respectively),linked by a linker sequence (L) in several possible orientations, forexample, but not limited to, the following Vα-L-Vβ, Vβ-L-Vα, Vα-Ca-L-Vβor Vβ-Cβ-L-Vα.

A number of papers describe the production of TCR heterodimers whichinclude the native disulphide bridge which connects the respectivesubunits. However, although such TCRs can be recognised by TCR-specificantibodies, none have been shown to recognise its native ligand atanything other than relatively high concentrations and/or were notstable.

In WO 03/020763 a soluble TCR is described which is correctly folded sothat it is capable of recognising its native ligand, is stable over aperiod of-time, and can be produced in reasonable quantities. This TCRcomprises a TCR α chain extracellular domain dimerised to a TCR β chainextracellular domain respectively, by means of an inter-chain disulfidebond between cysteines introduced into the constant regions of therespective chains.

Specific pMHC binding partners, ie antibodies specific for pMHCepitopes, and TCRs of both the heterodimeric and single chain type, havebeen proposed as targeting vectors for the delivery of therapeuticagents to antigen presenting cells. For that purpose, the therapeuticagent is required to be associated with the pMHC-binding partner in someway. Therapeutic agents which have been suggested for such targeteddelivery in association with pMHC-binding partners include antibodies(see for example: Mosquera L A. et al., J Immunol (2005) 174 (7):4381-8), cytokines (see for example: Belmont H J. et al., Clin Immunol(2006) 121 (1): 29-39; Wen J. et al. Cancer Immunol Immunother (2008) 57(12): 1781-94), and cytotoxic agents. Where the therapeutic agent is apolypeptide, the means of association with the pMHC binding partner maybe by peptidic fusion, either direct fusion or fusion via a linkersequence, to the pMHC binding partner. In those cases, there areessentially only two fusion possibilities. In the case of single chainantibodies or TCRs, fusion can in principle be at the C- or N-terminusof the TCR chain; In the case of heterodimeric antibodies or TCRs, thefusion can in principle be at the C- or N-terminus of either chain. Inpractice however, it appears that all known examples of pMHC bindingpartner-therapeutic agent fusions have been with the therapeutic agentfused to the C-terminus (see for example: Mosquera L A. et al., JImmunol (2005) 174 (7): 4381-8; Belmont H J. et al., Clin Immunol (2006)121 (1): 29-39; Wen J. et al., Cancer Immunol Immunother (2008) 57 (12):1781-94). This is because the functionality of an antibody or TCR,whether single chain or heterodimeric, depends on correct folding andorientation of the variable regions. Fusion of the therapeutic agent tothe N-terminus of the pMHC binding partner places it ahead of one of thevariable regions, and there has been an assumption in the art that thetherapeutic agent located at the N-terminus will interfere with bindingof the antibody or TCR to the pMHC complex, thereby reducing its bindingefficiency.

SUMMARY OF THE INVENTION

Contrary to that assumption in the art, it has now been found thatbifunctional molecules wherein an immune effector part is fused to theN-terminus of a pMHC-binding partner are more effective in theirinduction of the relevant immune response that the correspondingconstruct wherein the fusion is to the C-terminus of the pMHC-bindingpartner. This enhanced immune response of the N-fused construct achieveddespite the similar pMHC binding affinities of the N-and C-fusedversion.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows the amino acid sequence of the alpha chain of an NY-ESOTCR, in which C162 (using the numbering of SEQ ID No: 1) replaces T48 ofits TRAC constant region.

FIG. 2 shows the amino acid sequence of the beta chain NY ESO-TCR, inwhich C170 (using the numbering of SEQ ID No: 2) replaces S57 of itsTRBC2 constant region.

FIG. 3 shows the amino acid sequence of an anti CD3 UCHT-1 scFvantibody, with its intralinker sequence underlined.

FIG. 4 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Nterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L1 namelyGGEGS (SEQ ID No: 4).

FIG. 5 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Nterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L2 namelyAHHSEDPSSKAPKAP (SEQ ID No: 5).

FIG. 6 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Nterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L3 namelyGGEGGGSEGGGS (SEQ ID No: 6).

FIG. 7 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Cterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L4, whichin this case is the single amino acid, S.

FIG. 8 shows the amino acid sequence of the alpha chain of a TCR havingthe property of binding to a murine insulin-derived peptide, LYLVCGERG(SEQ ID NO: 8), presented by the murine H-2K^(d) complex,LYLVCGERG-H-2K^(d) (“LYLVCGERG” disclosed as SEQ ID NO: 8), in whichC158 (using the numbering of SEQ ID No: 7) replaces T48 of its TRACconstant region.

FIG. 9 shows the amino acid sequence of the beta chain of the same TCRwhich binds the murine LYLVCGERG-H-2Kd complex (“LYLVCGERG” disclosed asSEQ ID NO: 8), in which C171 (using the numbering of SEQ ID No: 9)replaces S57 of its TRBC2 constant region.

FIG. 10 shows the amino acid sequence of a murine IL-4 polypeptiderepresented by SEQ ID No: 10.

FIG. 11 shows SEQ ID No: 11 which is the amino acid sequence of a murineIL-13 polypeptide.

FIG. 12 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having murine IL-4 SEQ ID No: 10 fused at the N terminus ofthe TCR β chain SEQ ID No: 9 via the linker sequence L5, namely GGEGGGP(SEQ ID No: 12).

FIG. 13 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having the murine IL-4 (SEQ ID No: 10) fused at the Cterminus of the TCR β chain (SEQ ID No: 9) via the linker sequence L6,namely GSGGP (SEQ ID No: 13).

FIG. 14 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having murine IL-13 (SEQ ID No: 11) fused at the N terminusof the TCR β chain (SEQ ID No: 9) via the linker sequence L5, namelyGGEGGGP (SEQ ID No: 12).

FIG. 15 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having murine IL-13 (SEQ ID No: 11) fused at the C terminusof the TCR β chain (SEQ ID No: 9) via the linker sequence L6, namelyGSGGP (SEQ ID No: 13).

FIG. 16 shows the amino acid sequence (SEQ ID No:14) of the beta chainof FIG. 2 with the N-terminus of an anti-CD3 scFv fused to theC-terminus of the TCR beta chain via another peptide linker sequence(underlined).

FIG. 17 shows the amino acid sequence (SEQ ID No:15) of the beta chainof FIG. 2 with the C-terminus of an anti-CD3 scFv fused to theN-terminus of the TCR beta chain via the same peptide linker sequence asin SEQ ID No 14 (again underlined).

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the present invention provides a bifunctional moleculecomprising a polypeptide binding partner specific for a given pMHCepitope, and an immune effector polypeptide, the N-terminus of the pMHCbinding partner being linked to the C-terminus of the immune effectorpolypeptide, PROVIDED THAT the said polypeptide binding partner is not aT-cell receptor comprising the alpha chain SEQ ID No: 7 and the betachain SEQ ID No: 9.

As mentioned, the polypeptide pMHC binding partner may be an antibody ora TCR. Thus in one embodiment of the invention the pMHC binding partneris a heterodimeric TCR polypeptide pair, or a single chain TCRpolypeptide, and the N-terminus of the α or β chain of the heterodimericTCR polypeptide pair, or the N-terminus of the scTCR polypeptide, islinked to a C-terminal amino acid of the immune effector polypeptide.

Linkage of the pMHC binding partner and the immune effector polypeptidemay be direct, or indirect via linker sequence. Linker sequences areusually flexible, in that they are made up of amino acids such asglycine, alanine and serine which do not have bulky side chains likelyto restrict flexibility. Usable or optimum lengths of linker sequencesare easily determined in the case of any given pMHC bindingpartner-immune effector construct. Often the linker sequence will byless than about 12, such as less that 10, or from 5-10 amino acids inlength.

In some embodiments of the invention the pMHC binding partner is aheterodimeric αβ TCR polypeptide pair wherein the α and β polypeptideseach have TCR variable and constant regions, but lack TCR transmembraneand cytoplasmic regions. The TCR part in such cases is soluble. Inparticularly preferred bifunctional molecules of this type, a non-nativedisulfide bond between residues of the constant regions of TCR α and βpolypeptides is present. In particular the constant regions of the α andβ polypeptides may be linked by a disulfide bond between cysteineresidues substituted for Thr 48 of exon 1 of TRAC1 and Ser 57 of exon 1of TRBC1 or TRBC2, or by the native disulfide bond between Cys4 of exon2 of TRAC*01 and Cys2 of exon 2 of TRBC1 or TRBC2.

In other embodiments of the invention, the pMHC binding partner is asingle chain αβ TCR polypeptide of the Vα-L-Vβ, Vαβ-L-Vα, Vα-Cα-L-Vβ, orVα-L-Vβ-Cβ type wherein Vα and Vβ are TCR α and β variable regionsrespectively, Cα and Cβ α are TCR α and β constant regions respectively,and L is a linker sequence.

Immune effector polypeptides are known. They are molecules which induceor stimulate an immune response, through direct or indirect activationof the humoural or cellular arm of the immune system, such as byactivation of T-cells. Examples include: IL-1, IL-1α, IL-3, IL-4, IL-5,IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β,IFN-γ, TNFα, Anti-CD2 antibody, Anti-CD3 antibody, Anti-CD4 antibody,Anti-CD8 antibody, Anti-CD44 antibody, Anti-CD45RA antibody, Anti-CD45RBantibody, Anti-CD45RO antibody, Anti-CD49a antibody, Anti-CD49bantibody, Anti-CD49c antibody, Anti-CD49d antibody, Anti-CD49e antibody,Anti-CD49f antibody, Anti-CD16 antibody, Anti-CD28 antibody, Anti-IL-2Rantibodies, Viral proteins and peptides, and Bacterial proteins orpeptides. Where the immune effector polypeptide is an antibody it may bescFv antibody, one such being an anti-CD3 scFv. Examples of anti-CD3antibodies include but are not limited to OKT3, UCHT-1, BMA031 and 12F6.

The principles of the invention are illustrated by the followingExamples.

Example A. Preparation of Soluble αβ TCRs Having Effector PolypeptidesFused to the C- or N-Terminus of the TCR β Chain

-   -   A1. Soluble NY-ESO TCR with Anti-CD3 Antibody as Effector        Polypeptide

The soluble NY-ESO TCR of this example has the property of binding tothe SLLMWITQV peptide (SEQ ID NO: 16) when presented on an HLA-A2molecule.

SEQ ID No: 1 (FIG. 1) is the amino acid sequence of the alpha chain ofan NY-ESO TCR, in which C162 (using the numbering of SEQ ID No: 1)replaces T48 of its TRAC constant region.

SEQ ID No: 2 (FIG. 2) is the amino acid sequence of the beta chain NYESO-TCR, in which C170 (using the numbering of SEQ ID No: 2) replacesS57 of its TRBC2 constant region.

SEQ ID No: 3 (FIG. 3) is the amino acid sequence of an anti CD3 UCHT-1scFv antibody, with its intralinker sequence underlined.

FIG. 4 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Nterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L1 namelyGGEGS (SEQ ID No: 4).

SEQ ID No: 14 (FIG. 16) is the amino acid sequence of the beta chain ofFIG. 2 with the N-terminus of an anti-CD3 scFv fused to the C-terminusof the TCR beta chain via another peptide linker sequence (underlined).

SEQ ID No: 15 (FIG. 17) is the amino acid sequence of the beta chain ofFIG. 2 with the C-terminus of an anti-CD3 scFv fused to the N-terminusof the TCR beta chain via the same peptide linker sequence as in SEQ IDNo 14 (again underlined).

The construct of FIG. 4 was prepared as follows:

Ligation

Synthetic genes encoding (a) the TCR α chain SEQ ID No: 1 and (b) thefusion sequence of SEQ ID No: 2 and SEQ ID No: 3, were separatelyligated into pGMT7-based expression plasmids, which contain the T7promoter for high level expression in E. coli strain BL21-DE3(pLysS)(Pan et al., Biotechniques (2000) 29 (6): 1234-8.

Expression

The expression plasmids were transformed separately into E. coli strainBL21 (DES) Rosetta pLysS, and single ampicillin-resistant colonies weregrown at 37° C. in TYP (ampicillin 100 μg/ml) medium to OD₆₀₀ of˜0.6-0.8 before inducing protein express with 0.5 mM IPTG. Cells wereharvested three hours post-induction by centrifugation for 30 minutes at4000 rpm in a Beckman J-6B. Cell pellets were lysed with 25 ml BugBuster (NovaGen) in the presence of MgCl₂ and DNase. Inclusion bodypellets were recovered by centrifugation for 30 minutes at 13000 rpm ina Beckman J2-21 centrifuge. Three detergent washes were then carried outto remove cell debris and membrane components. Each time the inclusionbody pellet was homogenised in a Triton buffer (50 mM Tris-HCl pH 8.0,0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA,) before being pelleted bycentrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergentand salt was then removed by a similar wash in the following buffer: 50mM Tris-HCl pH 8.0, 1 mM NaEDTA. Finally, the inclusion bodies weredivided into 30 mg aliquots and frozen at −70° C.

Refolding

Approximately 20 mg of TCR α chain and 40 mg of scFv-TCR β chainsolubilised inclusion bodies were thawed from frozen stocks, dilutedinto 20 ml of a guanidine solution (6 M Guanidine-hydrochloride, 50 mMTris HCl pH 8.1, 100 m NaCl, 10 mM EDTA, 20 mM OTT), and incubated in a37° C. water bath for 30 min-1 hr to ensure complete chainde-naturation. The guanidine solution containing fully reduced andenatured TCR chains was then injected into 1 litre of the followingrefolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride (to final concentrations of 16 mM and 1.8 mM,respectively)) were added approximately 5 minutes before addition of thedenatured TCR α and scFv-TCR β chains. The solution was left for ˜30minutes. The refolded scFv-TCR was dialysed in dialysis tubing cellulosemembrane (Sigma-Aldrich; Product No. D9402) against 10 L H₂O for 18-20hours. After this time, the dialysis buffer was changed twice to fresh10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5° C.±3° C. foranother ˜8 hours. Soluble and correctly folded scFv-TCR was separatedfrom misfolded, degradation products a impurities by a 3-steppurification method as described below. The second purification step caneither be an ion exchange chromatography or an affinity chromatography,depending on the pl of the soluble. anti-CD3 scFv-TCR fusion.

First Purification Step

The dialysed refold (in 10 mM Tris pH8.1) was loaded onto a POROS 50HQanion exchange column and the bound protein eluted with a gradient of0-500 mM NaCl over 6 column volumes using an Akta purifier (GEHealthcare). Peak fractions (eluting at a conductivity ˜20 mS/cm) werestored at 4° C. Peak fractions were analysed by Instant Blue Stain(Novexin) stained SDS-PAGE before being pooled.

Second Purification Step Ion Exchange Chromatography Cation ExchangePurification:

The anion exchange pooled fractions were buffer exchanged by dilutionwith 20 mM MES pH6-6.5, depending on the pl of the scFv-TCR fusion. Thesoluble and correctly folded scFv-TCR was separated from misfolded,degradation products and impurities by loading the diluted pooledfractions (in 20 mM MES pH6-6.5) onto a POROS 50HS cation exchangecolumn and eluting bound protein with a gradient of 0-500 mM NaCl over 6column volumes using an Akta purifier (GE Healthcare). Peak fractions(eluting at a conductivity ˜10 mS/cm) were stored at 4° C.

Alternatively, ion exchange purification using hydroxyapatite matrix canbe used a explained below.

Hydroxyapatite Chromatography:

The anion exchange pooled fractions were buffer exchanged by dilutionwith 10 mM NaH₂PO₄ pH6.0. The soluble and correctly folded scFv-TCR wasseparated from misfolded, degradation products and impurities by loadingthe diluted pooled fractions (in 10 mM NaH₂PO₄ pH6.0) onto ahydroxyapatite column and eluting bound protein with a gradient of10-500 mM NaH₂PO₄/1M NaCl over 6 column volumes using an Akta purifier(GE Healthcare). Peak fractions (eluting at a conductivity ˜20 mS/cm)were stored at 4° C.

Affinity Chromatography

For some scFv-TCR fusions with a pl close to 6-6.5, the ion exchangestep cannot be used but can be replaced by an affinity chromatographystep. The protein L affinity chromatography column (Pierce, productnumber 89928) isolates and purifies certain immunoglobulin classes viatheir kappa light chains. Protein L can also binds sing chain variablefragments (scFv). The anion exchange pooled fractions were bufferexchanged by dilution with PBS/0.02% sodium azide. The soluble andcorrectly folded scFv-TCR was separated from misfolded, degradationproducts and impurities by loading the diluted pooled fractions onto aProtein L column and eluting bound protein with a gradient of 0-25 mMGlycine pH2.3/0.02% sodium azide over 3 column volumes using an Aktapurifier (GE Healthcare). The scFv-TCR eluted very late in gradient andthe pH of the eluted fractions was neutralized by addition of Tris pH8 1(100 mM Tris pH8.1 final concentration). The peak fractions were storedat 4° C.

Final Purification Step

Peak fractions from second purification step were analysed by InstantBlue Stain (Novexin) stained SDS-PAGE before being pooled. The pooledfractions were then concentrated for the final purification step, whenthe soluble scFv-TCR was purified and characterised using a Superdex5200 gel filtration column (GE Healthcare) pre-equilibrated in PBSbuffer (Sigma). The peak eluting at a relative molecular weight ofapproximately 78 kDa was analysed by Instant Blue Stain (Novexin)stained SDS-PAGE before being pooled. In a similar manner to thatdescribed for the construct of FIG. 4, the constructs o FIGS. 5, 6 and 7were prepared:

FIG. 5 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Nterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L2 namelyAHHSEDPSSKAPKAP (SEQ ID No: 5).

FIG. 6 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Nterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L3 namelyGGEGGGSEGGGS (SEQ ID No: 6).

FIG. 7 shows in block diagram form the structure of a soluble NY-ESO αβTCR having the α chain SEQ ID No: 1 and the β chain SEQ ID No: 2, andhaving the anti CD3 UCHT-1 scFv antibody SEQ ID No: 3 fused at the Cterminus of the TCR β chain SEQ ID No: 2 via a linker sequence L4 whichin this case is single amino acid S.

In a similar manner to that described for the constructs of FIGS. 4, 5,6 and 7, the fusion proteins having the TCR α chain SEQ ID No: 1 and theTCR β chain-anti-C scFv SEQ ID No: 14, where the anti-CD3 scFv is fusedto the C-terminus of the TCR beta chain, or the TCR α chain SEQ ID No: 1and the TCR β chain-anti-CD3 scFv SEQ ID No: 15, where the anti-CD3 scFvis fused to the N-terminus of the TCR beta chain, were prepared.

A2. Soluble Chimeric TCR with Cytokines as Effector Polypeptides

SEQ ID No: 7 (FIG. 8) is the amino acid sequence of the alpha chain of aTCR having the property of binding to a murine insulin-derived peptide,LYLVCGERG (SEQ ID NO: 8), presented by the murine H-2K^(d) complex.(LYLVCGERG-H-2K^(d) (“LYLVCGERG” disclosed as SEQ ID NO: 8)), in whichC158 (using the numbering of SEQ ID No: 7) replaces T48 of its TRACconstant region.

SEQ ID No: 9 (FIG. 9) is the amino acid sequence of the beta chain ofthe same TCR which binds the murine LYLVCGERG-H-2K^(d) complex(“LYLVCGERG” disclosed as SEQ ID NO: 8), in which C171 (using thenumbering of SEQ ID No: 9) replaces S57 of its TRBC2 constant region.

The SEQ ID No: 7 and 9 TCR is a chimeric TCR consisting of an alpha anda beta TCR chain, each comprising a murine variable region and a humanconstant region. The chimeric version of the TCR was constructed toimprove refolding problems encountered with the fully murine TCR; andthe chimeric TCR was shown to have the same affinity as the murine TCRfor the murine insulin-derived peptide-murine H-2K^(d) complex.

SEQ ID No: 10 (FIG. 10) is the amino acid sequence of a murine IL-4polypeptide.

SEQ ID No: 11 (FIG. 11) is the amino acid sequence of a murine IL-13polypeptide.

FIG. 12 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having murine IL-4 SEQ ID No: 10 fused at the N terminus ofthe TCR β chain SEQ ID No: 9 via the linker sequence L5, namely GGEGGGP(SEQ ID No: 12).

FIG. 13 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having the murine IL-4 SEQ ID No: 10 fused at the C terminusof the TCR β chain SEQ ID No: 9 via the linker sequence L6, namely GSGGP(SEQ ID No: 13).

FIG. 14 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having murine IL-13 SEQ ID No: 11 fused at the N terminus ofthe TCR β chain SEQ ID No: 9 via the linker sequence L5, namely GGEGGGP(SEQ ID No: 12).

FIG. 15 shows in block diagram form the structure of a soluble chimericinsulin αβ TCR having the α chain SEQ ID No: 7 and the β chain SEQ IDNo: 9, and having murine IL-13 SEQ ID No: 11 fused at the C terminus ofthe TCR β chain SEQ ID No: 9 via the linker sequence L6, namely GSGGP(SEQ ID No: 13).

The constructs of FIGS. 12-15 were prepared as follows.

Ligation

Synthetic genes encoding (a) the TCR α chain SEQ ID No: 7 and (b) thefusion sequence of SEQ ID No: 9 and SEQ ID No: 10 or 11, were separatelyligated into pGMT7-based expression plasmids, which contain the T7promoter for high level expression in E. coli strain BL21-DE3(pLysS)(Pan et al., Biotechniques (2000) 29 1234-8).

Expression

The expression plasmids containing the TCR α-chain and cytokine-β-chainrespectively were transformed separately into E. coli strain BL21 (DE3)Rosetta pLysS, and single ampicillin-resistant colonies were grown at37° C. in TYP (ampicillin 100 μg/ml) medium to OD₆₀₀ of ˜0.6-0.8 beforeinducing protein expression with 0.5 mM IPTG. Cells were harvested threehours post-induction by centrifugation for 30 minutes at 4000 rpm in aBeckman J-6B. Cell pellets were lysed with 25 ml Bug Buster (NovaGen) inthe presence of MgCl₂ and DNase. Inclusion body pellets were recoveredby centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21centrifuge. Three detergent washes were then carried out to remove celldebris and membrane components. Each time the inclusion body pellet washomogenised in a Triton buffer (50 mM Tris-HCl pH 8.0, 0.5% Triton-X100,200 mM NaCl, 10 mM NaEDTA,) before being pelleted by centrifugation for15 minutes at 13000 rpm in a Beckman J2-21. Detergent and salt was thenremoved by a similar wash in the following buffer: 50 mM Tris-HCl pH8.0, 1 mM NaEDTA. Finally, the inclusion bodies were divided into 30 mgaliquots and frozen at −70° C. Inclusion body protein yield wasquantified by solubilising with 6M guanidine-HCl and an OD measurementwas taken on a Hitachi U-2001 Spectrophotometer. The proteinconcentration was the calculated using the theoretical extinctioncoefficient.

Refolding

Approximately 20 mg of TCR α chain and 40 mg of cytokine-TCR β chainsolubilise inclusion bodies were thawed from frozen stocks and dilutedinto 20 ml of a guanidine solution (6 M Guanidine-hydrochloride, 50 mMTris HCl pH 8.1, 100 m NaCl, 10 mM EDTA, 10 mM DTT), and incubated in a37° C. water bath for 30 min-1 hr to ensure complete chainde-naturation. The guanidine solution containing fully reduced anddenatured TCR chains was then injected into 1 litre of cold (5-10° C.)refolding buffer: 100 mM Tris pH 8.1, 400 mM L-Arginine, 2 mM EDTA, 5MUrea. The redox couple (cysteamine hydrochloride and cystaminedihydrochloride (to final concentrations 10 mM and 2.5 mM,respectively)) were added approximately 5 minutes before addition of thedenatured TCR α and cytokine-TCR β chains. The solution was left for ˜30minutes. The refolded cytokine-TCR was dialysed in dialysis tubingcellulose membrane (Sigma-Aldrich; Product No. 09402) against 10 L H₂Ofor 18-20 hours. After this time, the dialysis buffer was changed twiceto fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5° C.±3°C. for another ˜8 hours.

Purification

Soluble cytokine-TCR fusion was separated from degradation products andimpurities by a 3-step purification method at RT as described below.

First Purification Step

The dialysed refold was filtered using a Sartopore 0.2 μm capsule(Sartorius) prior to column purification. Filtered refold was loadedonto a POROS 50HQ anion exchange column and the bound protein elutedwith a linear gradient of 0-500 mM NaCl over 6 column volumes using anAkta purifier (GE Healthcare). Peak fractions eluting at 250 mM NaCl,consisting of correctly folded protein, were stored at 4° C. Peakfractions were analysed by Instant Blue Stain (Novexin) stained SDS-PAGEbefore being pooled.

Second Purification Step

Pooled fractions containing soluble cytokine-TCR were mixed with anequivalent volume of 50 mM Tris/1M (NH₄)₂SO₄ pH 8 to give a finalconcentration of 0.5 M (NH₄)₂SO₄ and a conductivity of 75-80 mS/cm atRT. The soluble cytokine-TCR were separated from degradation productsand impurities by loading this sample onto pre-equilibrated (50 mMTris/0.5M (NH₄)₂SO₄ pH 8) butyl hydrophobic interaction column (5 mlHiTrap GE Healthcare) and collecting the flow through using an Aktapurifier (GE Healthcare). Flow through sample containing solublecytokine-TCR was analysed by Instant Blue Stain (Novexin) stainedSDS-PAGE before being pooled and stored at 4° C.

Final Purification Step

Pooled fractions were diluted with an equivalent volume of 10 mM TrispH8 and concentrated to 10 ml (concentration of ≤3 mg/ml). The solublecytokine-TCR as purified using a Superdex S200 gel filtration column (GEHealthcare) pre-equilibrated in PBS buffer (Sigma). The peak eluting ata relative molecular weight of approximately 63 kDa was analysed byInstant Blue Stain (Novexin) stained SDS-PAGE before being pooled.

Example B. Properties of Soluble αβ TCRs Having Effector PolypeptidesFused to the C- or N-Terminus of the TCR β Chain

B1. Soluble NY-ESO TCR with Anti-CD3 Antibody as Effector Polypeptide

a. Redirection and Activation of CD8⁺ T Cells by the Soluble NY-ESO TCRFused to an Anti-CD3 Antibody Against NY-ESO Peptide-Presenting Cells

The following assay was carried out to demonstrate the activation ofcytotoxic T lymphocytes (CTLs) by an anti-CD3 scFv-TCR fusion viaspecific peptide-MHC complex. IFN-γ production, as measured using theELISPOT assay, was used as a read-out for cytotoxic T lymphocyte (CTL)activation and the evaluation of the potency of the anti-CD3 scFvportion of the fusion.

Reagents

Assay media: 10% FCS (Gibco, Cat#2011-09), 88% RPMI 1640 (Gibco,Cat#42401), 1% glutamine (Gibco Cat#25030) and 1%penicillin/streptomycin (Gibco Cat#15070-063).

Peptide: (SLLMWITQV (SEQ ID NO: 16)) initially dissolved in DMSO (Sigma,cat#D2650) at 4 mg/ml and frozen. T2 cells were pulsed with thedescribed peptide and used as target cells.

Wash buffer: 0.01 M PBS/0.05% Tween 20

PBS (Gibco Cat#10010)

The Human IFNγ ELISPOT PVDF-Enzymatic kit (Diaclone, France;Cat#856.051.020) contains all other reagents required. (Capture anddetection antibodies, skimmed milk powder, BSA, streptavidin-alkalinephosphatase and BCIP/NBT solution as well as the Human IFN-γ PVDFELISPOT 96 well plates)

Method Target Cell Preparation

The target cells used in this method were either (1) naturalepitope-presenting cell (such as Mel624 or Mel526 cells) or (2) T2 cellspulsed with the peptide of interest, described in the reagents section.Sufficient target cells (50 000 cells/well) were washed bycentrifugation three times at 1200 rpm, 10 min in a Megafuge 1.0(Heraeus). Cells were then re-suspended in assay media at 10⁶ cells/ml.

Effector Cell Preparation

The effector cells (T cells) used in this method were either CD8+ Tcells (obtained by negative selection (using the CD8 Negative IsolationKit, Dynal, Cat#113.19) from PBL), T cells from an EBV cell line orPBMCs. Effector cells were defrosted and placed in assay media prior towashing by centrifugation at 1200 rpm, 10 min in a Megafuge 1.0(Heraeus). Cells were then re-suspended in assay media at a 4× the finalrequired concentration.

Reagent/Test Compound Preparation

Varying concentrations of test compounds (the TCR-anti-CD3 fusions; from10 nM to 0.03 pM) were prepared by dilution into assay media to give 4λfinal concentration.

ELISPOTs

Plates were prepared as follows: 100 μl anti-1FN-y capture antibody wasdiluted in 10 ml sterile PBS per plate. 100 μl of the diluted captureantibody was then aliquoted into each well. The plates were thenincubated overnight at 4° C. Following incubation the plates were washed(programme 1, plate type 2, Ultrawash Plus 96-well plate washer; Dynex)to remove the capture antibody. Plates were then blocked by adding 100μl 2% skimmed milk in sterile PBS to each well and incubating the platesat room temperature for two hours. The skimmed milk was then washed fromthe plates (programme 1, plate type 2, Ultrawash Plus 96-well platewasher, Dynex) and any remaining wash buffer was removed by flicking andtapping the ELISPOT plates on a paper towel.

The constituents of the assay were then added to the ELISPOT plate inthe following order:

-   -   50 μl of target cells 10⁶ cells/ml (giving a total of 50 000        target cells/well)    -   50 μl of reagent (the anti-CD3 scFv-TCR fusions; varying        concentrations)    -   50 μl media (assay media)    -   50 μl effector cells (between 1000 and 50000 CD8⁺ cells/well;        between 500 and 1000 EBV cells/well; between 1000 and 50000        PBMC/well).

The plates were then incubated overnight (37° C./5% CO₂). The next daythe plates were washed three times (programme 1, plate type 2, UltrawashPlus 96-well plate washer, Dynex) with wash buffer and tapped on papertowel to rer1 love excess wash buffer. 100 50 μl primary detectionantibody was then added to each well. The primary detection antibody wasprepared by adding 550 μl of distilled water to a vial of detectionantibody supplied with the Diaclone kit. 100 μl of this solution wasthen diluted in 10 ml PBS/1% BSA (the volume required for a singleplate). Plates were then incubated at room temperature for at least 2 hrprior to being washed three times (programme 1, plate type 2, UltrawashPlus 96-well plate washer, Dynex) with wash buffer, excess wash bufferwas removed by tapping the plate on a paper towel. Secondary detectionwas performed by adding 100 μl of diluted streptavidin-Alkalinephosphatase to each well and incubating the plate at room temperaturefor 1 hour. The streptavidin-Alkaline phosphatase was prepared byaddition of 10 μl streptavidin-Alkaline phosphatase to 10 ml PBS/1% BSA(the volume required for a single plate). The plates were then washedthree times (programme 1, plate type 2, Ultrawash Plus 96-well platewasher, Dynex) with wash buffer and tapped on paper towel to removeexcess wash buffer. 100 μl of BCIP/NBT solution, as supplied with theDiaclone kit, was then added to each well. During development plateswere covered in foil and left for 5-15 min. Developing plates wereregularly checked for spots during this period to determine optimal timeto terminate the reaction. The plates were washed in a sink full of tapwater to terminate the development reaction, and shaken dry prior totheir disassembly into their three constituent parts. The plates werethen dried at 50° C. for 1 hr prior to counting the spots that haveformed on the membrane using an Immunospot Plate reader (CTL; CellularTechnology Limited).

Results

The anti-CD3 scFv-TCR fusion constructs of FIGS. 4-7 were tested byELISPOT assay (as described above). The number of ELISPOT spots observedin each well was plotted against the concentration of the test constructusing Prism (Graph Pad).

From these dose-response curves, the EC₅₀ values were determined (EC₅₀are determined at the concentration of anti-CD3 scFv-TCR fusion thatinduces 50% of the maximum response).

TABLE 1 Test Construct EC50 EC50 EC50 FIG. 7 C-terminal fusion 5.044e−9 1.864e−9  2.383e−9 FIG. 5 N-terminal fusion 8.502e−11 FIG. 4 N-terminalfusion 4.825e−11 FIG. 6 N-terminal fusion  3.95e−11

These results show that the N-fused constructs of FIGS. 4, 5 and 6 wereat least 2 fold more potent in their ability to activate cytotoxic Tlymphocytes than the C-fused construct of FIG. 7.

b. Redirection of CD8+ T Cells by the Soluble NY-ESO TCR Fused to anAnti-CD3 Antibody to Kill the IM9 EBV Transformed B Cell Line(Non-Radioactive Cytotoxicity Assay)

The following assay was carried out to demonstrate: the activation ofcytotoxic T lymphocytes (CTLs) by a TCR-anti-CD3 scFv fusion viaspecific peptide-MHC complex and the evaluation of the potency of theanti-CD3 scFv portion of the fusion to activate the CTLs to kill the IM9cells. This assay is a colorimetric alternative to ⁵¹Cr releasecytotoxicity assays and quantitatively measures lactate dehydrogenase(LDH) which is an enzyme that is released upon cell lysis. Released LDHin culture supernatants is measured with a 30-minute coupled enzymaticassay, which results in the conversion of a tetrazolium salt (INT) intoa red formazan product. The amount of colour formed is proportional tothe number of lysed cells. The absorbance data is collected using astandard 96-well plate reader at 490 nm.

Materials

CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega) (G1780) containsSubstrate Mix, Assay Buffer, Lysis Solution, and Stop Solution

Assay media: 10% FCS (heat-inactivated, Gibco, cat#10108-165), 88% RPMI1640 without phenol red (Invitrogen, cat#32404014), 1% glutamine, 200 mM(Invitrogen, cat#25030024), 1% penicillin/streptomycin (Invitrogencat#15070063)

Nunc microwell round bottom 96 well tissue culture plate (Nunc,cat#163320)

Nunc-Immuno plates Maxisorb (Nunc, cat#442404)

Method Target Cell Preparation

The targets cells used in this assay were the IM9 EBV transformed Bcell-line derived from a multiple myeloma patient (HLA-A2⁺ NY-ESO). TheMel526 melanoma cell line was used as a control and is HLA-A2⁺ NY-ESO.Target cells were prepared in assay medium: target cell concentrationwas adjusted to 2×10⁵ cells/ml to give 1×10⁴ cells/well in 50 μl.

Effector Cell Preparation

The effector cells used in this assay were CD8⁺ T cells. The effector totarget ratio used was 10:1 (2×10⁶ cells/ml to give 1×10⁵ cells/well in50 μl).

Reagent/Test Compound Preparation

Varying concentrations of the NY-ESO TCR-anti-CD3 fusions, having theTCR alpha chain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusionSEQ ID No: 14, or having the TCR alpha chain SEQ ID No: 1 and the TCRbeta chain-anti-CD3 scFv fusion SEQ ID No: 15, were prepared asdescribed in example A1 and prepared for this assay by dilution (10⁻¹³to 10⁻⁸M final concentration) into assay media.

Assay Preparation

The constituents of the assay were added to the plate in the followingorder:

50 μl of target cells, IM9 or Mel526 (prepared as explained previously),to each well

50 μl of reagent (prepared as explained previously) to each well.

50 μl of effector cells (prepared as explained previously) to each well

Several controls were prepared as explained below:

Effector spontaneous release: 50 μl effector cells alone.

Target cells spontaneous release: 50 μl target cells alone.

Target maximum release: 50 μl target cells plus 80 ug/ml digitonin atthe start of the assay to lyse cells

Assay medium control: 150 μl medium alone.

Experimental wells are prepared in triplicate and control wells induplicate in a final volume of 150 μl.The plate was centrifuged at 250×g for 4 minutes then incubated at 37°C. for 24 hours.

The plate was centrifuged at 250×g for 4 minutes. 37.5 μl of thesupernatant from each well of the assay plate was transferred to thecorresponding well of a flat-bottom 96 well Nunc Maxisorb plate. TheSubstrate Mix was reconstituted using Assay Buffer (12 ml). 37.5 μl ofthe reconstituted Substrate Mix was then added to each well of theplate. The plate was covered with aluminum foil and incubated at roomtemperature for 30 minutes. 37.5 μl of Stop Solution was added to eachwell the plate to stop the reaction. The absorbance at 490 nm wasrecorded on an ELIS plate reader within one hour after the addition ofStop Solution.

Calculation of Results

The average of absorbance values of the culture medium background wassubtracted from all absorbance values of Experimental, Target CellSpontaneous Release and Effector Cell Spontaneous Release and Targetmaximum release.

The corrected values obtained in the first two steps were used in thefollowing formula to compute percent cytotoxicity:

% cytotoxicity=100×(Experimental−Effector Spontaneous−TargetSpontaneous)/(Target Maximum Release−Target Spontaneous)

Results

The NY-ESO TCR-anti-CD3 scFv fusion constructs having (i) the TCR alphachain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ IDNo: 14 (C-terminal fusion) or (ii) the TCR alpha chain SEQ ID No: 1 andthe TCR beta chain anti-CD3 scFv fusion SEQ ID No: 15 (N-terminalfusion) were tested by LDH release assay (as described above). The %cytotoxicity observed in each well was plotted against the concentrationof the test construct using Prism (Graph Pad). From these dose-responsecurves, the EC50 values were determined (EC50 are determined at theconcentration of TCR fusion that induces 50% of the maximum response).

TABLE 2 Test Construct EC50 C-terminal fusion 1.2e⁻⁹  (SEQ ID No: 1 andSEQ ID No: 14) N-terminal fusion 3.2e⁻¹¹ (SEQ ID No: 1 and SEQ ID No:15)

These results show that the N-terminal fusion comprising the TCR alphachain SEQ ID No: 1 and the TCR beta chain-anti-CD3 scFv fusion SEQ IDNo: 15 was at least 2-fold more potent in its ability to redirectcytotoxic T lymphocytes to kill the target cells than the C-terminalfusion construct comprising the TCR alpha chain SEQ ID No: 1 and the TCRbeta chain-anti-CD3 scFv fusion SEQ ID No: 14.

B2. Soluble Chimeric TCR with Cytokines as Effector Polypeptides

a. Murine IL-4 Cytokine as Effector Polypeptide

The following assay was used to test the biological activity of thecytokine portion of the murine IL-4-TCR fusion constructs of FIGS.12-13. This is a bioassay using the murine cell line, CTLL-2, which aredependent on murine IL-4 for growth and are used here to demonstrate thebiological activity of the cytokine portion of a murine IL-4-TCR fusion.

Materials

CTLL-2 cells, Promega CellTiter-Glo® luminescent cell viability assay(Cat# G7572) including CellTiter-Glo® Buffer and CellTiter-Glo®Substrate (lyophilized) Assay media: RPMI supplemented with 10% heatinactivated foetal bovine serum (Gibco, cat#10108-165), 88% RPMI 1640(Gibco, cat#42401-018), 1% glutamine (Gibco, cat#25030-024), 1%penicillin/streptomycin (Gibco, cat#15070-063).

CTLL-2 cells were harvested, washed once in assay media (centrifuged at1200 rpm for 5 mins), counted, and viability was assessed using Trypanblue solution. If viability was less than 80% a ficoll gradient wasperformed to remove the dead cells (800×g for 15 mins with brake off).Cells were washed a further two times and the volume was adjusted togive 1×10⁵ cells/ml final. CTLL-2 cells were added to a Nunc whiteflat-bottomed 96-well plate (5000 cells/well), followed by 50 μltitrated concentrations of standard murine IL-4 (Peprotech), or murineIL-4-chimeric TCR fusion constructs of FIGS. 12 and 13 (7 points of 1 in10 dilutions, from 10⁻⁸ to 10¹⁴M). Controls included cells alone, assaymedia only, and cells with 200 U/ml Proleukin (Chiron). The plate wasincubated at 37° C., 5% CO2 overnight. Following the manufacturersinstructions, CellTiter-Glo reagent was thawed and added to plate (100μl per well). The plate was incubated for 10 minutes to stabilise theluminescent signal and then recorded using the luminescence reader. Thebackground signal (cells alone) was subtracted from the readings and agraph plotted in Prism (Graph Pad) so that the EC50's of the murineIL-4-TCR fusion constructs of FIGS. 12 and 13 can be compared with the‘free’ recombinant murine IL-4.

Results

TABLE 3 Test construct EC50 EC50 EC50 m-IL4 4.984e−13 3.767e−135.148e−13 FIG. 13 7.464e−12 C-term fusion FIG. 12 5.913e−13 8.897e−13N-term fusion

These results show that the N-fused construct of FIG. 12 was at least 2fold more potent in its ability to activate cell proliferation than theC-fused construct of FIG. 13.

b. Murine IL-13 Cytokine as Effector Polypeptide

The following assay was used to test the biological activity of thecytokine portion of the murine IL-13-TCR fusion constructs of FIGS.14-15.

This assay was carried out to demonstrate the activity of the cytokineportion from a cytokine-TCR fusion, i.e. the inhibition of theproduction of IL-1β by human monocytes. This assay can be used to testcytokine-TCR fusions where the cytokine is murine IL-13.

Materials

Monocytes Derived from Buffy Coats (Buffy Coats from NBS BristolTransfusion Service)

Dynal Dynabeads MyPure Monocyte Kit 2 for untouched human cells (113.35)Assay media: 10% foetal bovine serum (heat-inactivated, Gibco,cat#10108-165), 88% RPMI 1640 (Gibco, cat#42401-018), 1% glutamine(Gibco, cat#25030-024), 1% penicillin/streptomycin (Gibco,cat#15070-063)

Wash buffer: 0.01M PBS/0.05% Tween 20 (1 sachet of Phosphate bufferedsaline with Tween 20, pH7.4 from Sigma, cat# P-3563 dissolved in 1 Litredistilled water gives final composition 0.01M PBS, 0.138M NaCl, 0.0027MKCl, 0.05% Tween 20) PBS (Gibco, cat#10010-015).

HBS⁺S Ca⁺² an⁺d ⁺² Free (Gibco, cat#1018-165)

Cytokine Eli-pair ELISA kits: IL-1β (Diaclone cat# DC-851.610.020) thesekits contain all other reagents required i.e. capture antibody,detection biotinylated antibody, streptavidin-HRP, IL-1β standards,ready-to-use TMB. The following method is based on the instructionssupplied with each kit.

Nunc-Immuno plates Maxisorb (Nunc, cat#442404).

Nunc microwell round bottom 96 well tissue culture plate (Nunc,cat#163320)

BSA (Sigma, cat# A3059)

H2SO4 (Sigma cat# S1526)

Trypan blue (Sigma cat# T8154)

Lipopolysaccharides (LPS) derived from E. coli 0111:84 (Sigma, cat#L4391)

Recombinant murine IL-13 (Peprotech, cat#210-13) standard used whenmurine IL-13-TCR fusion reagents tested.

Monocyte Isolation

PBMCs were isolated from buffy coats: a buffy coat was diluted 1 in 2with HBSS (Ca²⁺ and Mg²⁺ free), diluted blood was layered ontolymphoprep (up to 35 ml blood over 15 ml lymphoprep) and centrifuged 15min at 800×g (room temp) with the brake off; cells at the interface wereremoved and washed four times with HBSS and centrifuged at 1200 rpm for10 min. After the final wash, cells were resuspended 50 ml assay mediacounted and viability was assessed using Trypan blue solution. DynalDynabeads MyPure Monocyte Kit 2 was used to isolate the monocytes. ThePBMC were resuspended in PBS/0.1% BSA in 100 μl buffer per 10⁷ cells, 20μl of Blocking Reagent per 10⁷ cells and 20 μl Antibody Mix per 10⁷cells were added and cells were incubated for 20 min at 4° C. The cellswere washed and resuspended in 0.9 ml PBS/0.1% BSA per 10⁷ cells.Pre-washed beads were added (100 μl per 10⁷ cells), mixed and incubatedfor a further 15 min at 20° C. with gentle rotation. Rose wereresuspended by careful pipetting and 1 ml PBS/0.1% BSA per 10⁷ cellswere added. The tube was placed in the Dynal magnet for 2 minutes.Supernatant containing negatively isolated cells was transferred to afresh tube and counted. Cells were either used immediately or frozendown in 90% FCS/10% DMSO for future use.

Cell Assay Preparation

The ELISA plate was coated with 100 μl/well IL-1β capture antibody inPBS and left at 4° C. overnight. Monocytes were thawed, washed twice inassay media and resuspended at 5×10⁵ cells/ml. The monocytes were platedout into a round bottomed 96 well plate (100 μl per well, i.e. 5×10⁴ perwell). LPS, Peprotech recombinant cytokine and test cytokine-TCR fusionproteins were prepared by dilution into assay media to give 4λ finalconcentration. LPS was added in each well (long/ml final) followed by 50μl of titrated concentrations (6 points of 1 in 10 serial dilutions) ofPeprotech recombinant IL-13 (10⁻⁸ to 10⁻¹³M final) or test cytokine-TCRfusion proteins (10⁻⁷ to 10⁻¹³M final) in triplicate wells. The platewas incubated at 37° C., 5% CO2 overnight.

IL-1β ELISA

The antibody coated IL-1β ELISA plate was washed three times in washbuffer and blocked with 250 μl PBS/5% BSA/well for at least 2 hours atroom temperature (or overnight at 4° C.). The ELISA plate was washedthree times in wash buffer and tapped dry. The IL-1β standards werediluted in PBS/1% BSA. The plate containing the cells was centrifuged at1200 rpm for 5 mins. The supernatant from each well was then transferredto the pre-coated IL-1β ELISA plate. 100 μl of cell supernatant (diluted1 in 3 with PBS/1% BSA) or standard were added to the relevant wells and50 μl detection antibody/well (dilution as per kit instructions) wereadded. The plate was incubated for 2 hours at room temperature. Plateswere washed three times in wash buffer. 100 μl of streptavidin-HRP wereadded per well (dilution as per kit instructions) and plates wereincubated at room temp for 20 min. Plates were washed three times inwash buffer. 100 μl of ready-to-use TMB per well were added and plateslet to develop for 5-20 min (depending on signal strength) in the dark(under foil). Reaction was stopped by adding 100 μl/well 1M H₂ SO₄.

Plates absorbance was read on microplate reader at 450 nm and areference filter set to 650 nm. The amount of inhibition for eachtitration point is calculated as a percentage of the sample containingmonocytes and LPS without cytokine—TCR fusion protein which gives themaximum signal thus producing a dose-response curve.

Results

TABLE 4 Test construct EC50 EC50 m-IL13 1.535e−10 9.534e−11 FIG. 15 6.21e−10 C-term fusion FIG. 14 1.493e−10 N-term fusion

These results show that the N-fused construct of FIG. 14 was at least 2fold more potent in its ability to inhibit the production of IL-1β byhuman monocytes than the C-fused construct of FIG. 15.

What is claimed is:
 1. A bifunctional molecule, comprising: a single chain T-Cell Receptor (scTCR), the scTCR comprising a TCR α or TCR β constant region, wherein the scTCR binds specifically to a given pMHC epitope, and an immune effector polypeptide comprising a single chain immunoglobulin (scFv) which specifically binds to CD3, wherein: the N-terminus of the scTCR is linked to the C-terminus of the scFv, and the bifunctional molecule is capable of binding to the given pMHC epitope and CD3, each on a different cell, and thus simultaneously binding to two different cells.
 2. The bifunctional molecule of claim 1, wherein the scFv comprises SEQ ID NO:
 3. 